All 19-carbon androgens derive from 21-carbon steroids via sequential 17[unreadable]- hydroxylase and 17,20-lyase activities of cytochrome P450c17 (CYP17A1). The complex chemistry of the 17,20-lyase reaction is selectively stimulated up to 10-fold by cytochrome b5 (b5). In contrast to the interactions of b5 with some other cytochromes P450, which apparently involve electron transfer from b5, our data argue that b5 allosterically activates the 17,20-lyase activity of CYP17A1. We have identified a specific region of b5 that is critical for stimulation of 17,20-lyase activity and have shown that the magnitude of this stimulation is substrate-dependent. We now propose to elucidate the mechanism of action of b5 on 17,20-lyase activity and to compare the mechanistic and structural features of the b5-CYP17A1 interaction with b5 action on other P450-mediated reactions. In Aim 1, we will determine the steric and electronic requirements of key residues on CYP17A1 and b5 necessary to stimulate 17,20-lyase activity. In Aim 2, we will deduce the microscopic steps of the 17,20-lyase reaction that are enhanced by b5 using rapid and pre-steady state kinetics experiments. In Aim 3, we will determine if the same surface of b5 important for stimulating 17,20-lyase activity also modulates the activities of other cytochromes P450 and whether the same mechanistic principles apply to these other reactions. In Aim 4, we will engineer soluble forms of b5 that also stimulate 17,20- lyase activity, setting the stage for future structural studies of the CYP17A1-b5 complex. We thus will systematically define the mechanism of action of b5 on CYP17A1, potentially identifying novel approaches for suppressing androgen production, by targeting the CYP17A1-b5 interaction. Diseases that involve androgen overproduction (such as polycystic ovary syndrome) and diseases that require androgens (such as prostate cancer) can be treated by inhibition of androgen synthesis. Currently available agents are suboptimal. By defining how the enzyme 17-hydroxylase/17,20-lyase makes androgens, we hope to establish new approaches to treating androgen-dependent human diseases. We will also determine if these principles apply to other similar enzymes that metabolize drugs. Inhibition of androgen synthesis is the current treatment of human diseases like prostate cancer and polycystic ovary syndrome. This approach is suboptimal. By defining how the enzyme responsible synthesizes these androgens, we hope to establish new approaches to treatment. Our study will apply to other enzymes that metabolize drugs.